AU597723B2 - Sodium scymnol sulphate isolated form shark tissues - Google Patents

Description

p a AU-A-78713/87 WORLD INEUU '0 0A NIZ O Intornt tio 11

PCT

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 International Publication Number: WO 88/ 012741 C07J 31/00, A61K 31/575 Al (43) International Publication Date: 25 February 1988 (25,02.88) (21) International Application Number: PCT/AU87/00281 (22) International Filing Date: (31) Priority Application Number: (32) Priority Date: (33) Priority Country: 21 August 1987 (21.08.87) PH 7614 21 August 1986 (21.08.86) (71) Applicant (for all designatedl States except US): BROADBENT, James, Meredyth [AU/AU]; Suite 2, 227 Burwood Road, Hawthorn, VIC 3122 (AU).

(72) Irnentors; and Inventors/Applicants (for US only) KOSUGE, Yoshliki [JP/JP]; KOSUGE, Takuo [JP/JP]; 3-4-18, Kamiashiarai, Shizuoka TSUJI, Kuniro JP/JP]: 2-1 1-17, Kamiashiarai, Shizuoka ISHIDA, Hitoshi [JP/ JP]; 200-16, Sena, Shizuoka (JP).

(74) Agents: SLATTERY, John, Michael et al.: Davies Collison, I Little Collins Street, Mvelbourne, VIC 3000 (AU), (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), DK, FR (European patent), GB (European patent), IT (European patent), JP, KR, LU (European patent), NL (European patent), SE (European patent), US, Published With: international search report.

A- P 3 1 MAR 1988 AUSTRALIAN 1 8 MAI? 1988 PATENT OFFICE ,(54)Title: ACTIVE PRINCIPLE ISOLATED FROM SHARK TISSUES

"OPH

1 ru du~~wen contai t he fIflments mDade under sect~lon 49.

and is Correct .prtl.

(57) Abstract IA compoun67 f general formula in substantially pure-form, wherein A is a cation.

also disclosed, together with compositions and methods of use thereof.

A method for preparation is Tj WO 88/01274 PCT/AU87/00281 "ACTIVE PRINCIPLE ISOLATED FROM SHARK TISSUES" This invention relates to the identification, isolation and preparation of an active principle by extraction from natural tissues, and in particular it relates to the identification, isolation and preparation of such an active principle by extraction from particular tissues of sharks.

In Japan, a preparation known as "deep-sea shark liver oil" has been used as a folk remedy for a long time. It is an oil prepared from shark's liver and is normally capsulated in soft capsules. The liver oil is said to be effective in treatment of many kinds of diseases, especially those which are related to the liver, such as hepatitis, nephritis, diabetes, etc. As well, when" used externally, it is widely recognised that the liver oil is effective in treatment of scalds, burns or other types of skin trouble, and also is ideal as an ingredient for cosmetics.

The present inventors have been studying this material for many years, and recently have discovered the unexpected fact that an active substance exists in the aqueous component of shark's liver rather than the oil soluble component. This fact was recognised from a comparison of the practical use of the liver oil and powder powder produced from the Ai I.I'"9 ~i r I ii' il~ill~ci;-li ~ii i 'll;(-ni.

I u rr~ -Lk PCT/AU87/00281 WO 88/01274 aqueous component of the liver by evaporation of the water. In comparative tests of 4 dosage of 900mg of the liver oil per day and 60mg of the powder per day, the latter gave a better clinical result than the former. Furthermore, where the liver oil as thoroughly washed with water, the resulti g oil showed almost no effect. These facts indicate that the active substance of deep-sea shark liver is not oil-soluble as previously believed, but is water-soluble.

According to the present invention, there is provided an active principl, which is isolated from an aqueous extract of the liver and/or gallbladder of a shark.

In a first aspect of the invention, there is provided a compound of the general formula I, in substantially pure form,

OH

CH OH

OH

SH20S03 wherein A is a cation, such as a sodium, potassium, calcium or ammonium ion, or an organic ine.

In other aspects, this invention provides a method for the preparation of a compound of general formula I in substantially pure form, together with -i_ :i i i ;~t-FI1:-ji~ ic; i I jA i J-1 WO 88/01274 PCT/AU87/00281 compositions for pharmaceutical, dietary or cosmetic purposes which comprise such a compound.

By using activity assays which are described in detail below, it has been shown that the active principle is water-soluble and does not exist in the oil-soluble component of shark's liver. These assays have been used in a series of tests to ascertain whether the active principle exists only in the liver.

All parts of the shark's body, such as the bones, meat, gallbladder, ovary, alimentary canal, etc., have been investigated, and it has been found that the gallbladder showed the same activities as liver in the assays. This result indicates that the active principle exists only in liver and gallbladder.

In general terms, the two bioassays referred to herein and used to identify sources of the active principle and to assess the degree of purity of an extract, are designed to identify characteristic pharmacological activities of the substance. In particular, the bioassays, designated as and (B) are based on the following activities: The active principle prevents liver trouble in mice caused by carbon tetrachloride.

The active principle increases the respiration rate in mice when a toxic substances such as nicotine is administered.

The present invention also provides a method for preparing an active principle as described above, which comprises the steps of preparing an aqueous extract of the liver and/or gallbladder of a shark, and isolating the active principle from the aqueous extract.

The following description sets out general procedures for isolation of the active principle from i^P n I-Q WO 88/01274 PCT/AU87/00281 4 the aqueous extract of the liver and/or gallbladder of a shark, involving the steps of extraction with polar organic solvents, adsorption on suitable adsorbents and/or chromatography techniques.

In order to determine whether the active principle is soluble in polar organic solvents, such as methanol, ethanol, acetone, etc., the powder obtained by freeze-drying of shark's bile was extracted with polar organic solvent, then the and assays were applied to both the soluble part and the insoluble part. Activity was seen only in the assays on the soluble portion, thus establishing that active principle is soluble in polar organic solvents.

In testing to determine whether the active principle can be isolated utilising adsorbents, many adsorbents were examined and it was found that the active principle can be adsorbed by ion exchange resins of basic anion exchange type, or by synthetic adsorbents such as XAD, HP-20, Sep-pak cl8, etc., or charcoal. This absorption test was performed by extracting shark's liver and/or gallbladder with water. Each adsorbent under test was added to the extract and left to stand overnight. The mixture was then filtered and each filtrate tested for activity by the and assays. The results indicate that the active substance is adsorbed by those adsorbents mentioned above. The active principle may be recovered from the adsorbent resins by extraction with acid, alkali or salts, and from the synthetic adsorbents and charcoal by extraction with polar orgeri.c solvents.

SFurther purification of the active principle is achieved by chromatography, for example in a silica column, Sephadex LH-20 column, or by preparative TLC 4 WO 88/01274 PCT/AU87/00281 (thin layer chromatography) or HPLC (high performance liquid chromatography), etc. Each method gave satisfactory results, but HPLC gave the best purification. The active principle as isolated by HPLC was quite pure because it gave very sharp single peak and also gave a single spot of approximate representative Rf value of 0.36 on TLC. The active principle in its purified form is a white powder of melting point of 140 0

C.

Testing of the purified active principle by vanillin sulfuric acid gave a purple colour, indicating that it contains bile acid or bile alcohol in its structure. It has already been found that the bile of sharks contains a bile alcohol named scymnol.

After partial acetylation of the active principle with acetic anhydride, followed by treatment of the crude product with dry dioxan-trichloroacetic acid for several days, scymnol was identified from the reaction mixture. The result indicated that the active principle is a scymnol derivative. It was the first isolation of the pure scymnol derivative contained in bile of shark, as the active principle.

A preferred procedure for isolation of the active principle from the lyophilized bile of Rhizoprinodon acutus (obtained by homogenization and freeze-drying of gall-bladders), is set out in the following chart: Th i i 1 1

I

1 i 1^ ;I1; WO 88/01274 PCT/AU87/00281 6 Lyophilized bile of Rhizoprionodon acutus extracted with 1. n-Hexane (100mlx3) 2. MeOH (100mlx3) Fraction 1.

2.

I(MeOH-extract) dissolved in Amberlite XAD-2 eluting with i. H 2 0 (400ml) ii. MeOH (400ml) Fraction II(MeOH-eluate) 1. dissolved in CHC 3-MeOH(1:1) 2. Sephadex LH-20 c.c. eluted with i. CHICl 3 -MeOH(:l1) (300ml) ii. MeOH (500ml) Fraction III HPLC: YMC-Pack A-324 (ODS) Colorless powder (compound I) As set out above, in this procedure the lyoophilized material is deflated with n-hexane, and then extracted with methanol. The concentrate thus obtained is applied to an Amberlite XAD-2 column in batches, using H 2 0, and ethanol as eluents. As the ethanol eluate contains the active principle (as determined by color reagent), this fraction is successively subjected to gel filtration on Sephadex with chloroform-methanol and methanol. The active principle is so effectively contained in the methanol eluate that its final purification is achieved by successive application of HPLC with a reverse phase column.

A

1 i- WO 88/01274 PCT/AU87/00281 7 It has been suggested that scymnol might be in the form of a sulphate ester, but no positive information has been published about the position of attachment of the sulphate ester, because scymnol has six hydroxyl groups where the sulphate ester group might be attached. The present scymnol derivative has never been isolated as a pure substance. The active powder as purified by HPLC was subjected to elementary analyses Results were anal: calcd for C 2 7

H

5 1 0 9

NS,

C;57.34, H;9.02, N;2.47, S;5.66. Found C;57.23, H;8.92, N;2.45, S;5.30. These results suggested that the active compound has ammonium sulphate ester in the structure. Nuclear magnetic resonance spectroscopy of the active powder showed the following properties.

H-NMR(in d 4 -MeOH) 6(ppm): 4.22(dd, 1H, J=4.5 and 10.0Hz), 4.11(dd, 1H, J=10.0 and 16.7Hz), 4.00(bs, 1H), 3.80(d, 1H, J=1.2Hz), 3.60-3.80(ma, 4H), 3.30-3.45(m, 1H) 0.72(s, 3H).

13 SC-NMR(in d 4 -MeOH) 6(ppm) 74.1(d), 72.9(d), 71.3(d), 69.1(d), 66.7(t), 61.2(t), 48.4(d), 47.8(d), 47.5(s), 43.1(d), 43.0(d), 41.0(d), 40.4(t), 37.0(d), 36.5(t), 35.9(s), 35.8(t), 33.3(t), 32.1(t), 31.2(t), 29.6(t), 28.8(t), 27.9(d), 24.3(t), 23.2(q), 18.1(q), 13.1(q).

13 C-NMR spectrum shows that the active compound has 27 carbon atoms made up of three methyl, 11 methylene, 11 methine and two tertiary carbons.

The signals at= low field (0.72-2.35) in 1H-NMR spectrum suggest that it seems to be a coprostane derivative. At the higher field in 1C-NMR spectrum, signals at 74.1(d), 72.9(d), 71.3(d) and 69.1(d) are lv t WO 88/01274 PCT/AU87/00281 8 assignable to the methine carbon with hydroxyl group.

And the two signals at 66.7(t) and 61.2(t) are ascribable to the O-substituted methylene carbon.

2DOCOSYONMR spectra and C-H-shift-COSY relationship indicate that these two carbons attach to a methine carbon and one of them with low chemical shift (66.7) has two unequivalent protons at 4.22(dd) and 4.14(dd)ppm in the 1H-NMR spectrum, which indicates that the active compound has the partial moiety of HOCH2-CH-CH 2 OR in the molecule. From the results of elementary analyses, R is -SO 3

NH..

From these NMR spectra and elementary analyses, the powder is characterised as 3a, 7a, 12a, 24 26-pentahydroxycoprostane-27-ammonium sulphate ester. The ammonium ion in the structure possibly came from the phosphate ammonium buffer used as mobile phase in HPLC, by replacement of a sodium ion. To verify this point, an active powder purified by XAD-2 and then by column chromatography on Sepadex LH-20 was subjected to atomic absorption spectrophotometry for sodium and to elementary analysis for nitrogen. The results were, calcd. for C 27

H

47 0 9 SNa, Na;4.03, N;0.00, found Na;3.57, N;0.02. The stereochemistry of the C-24 position in the structure was determined as 24R by X-ray crystallographic analysis of scymnol and the specific rotation of sodium scymnol sulphate is positive. Accordingly, it is concluded that the active principle isolated from shark is 24R-(+)-3a, 7a,12a,24,26-pentahydroxycrorostane-27-sodium sulphate ester.

S^ The sodium or ammonium ion in the sulphate ester is easily replaced by other metal ions such as potassium, calcium, etc., or by organic amine cations

S''

IS,

hh. I? or- I

-I

WO 88/01274 PCT/AU87/00281 such as amino acids, etc., by means of well known procedures.

The following Tables illustrate the activity of the aqueous extracts of this invention: TABLE I Bioassay(A) Bioassay(B) Dosage (Units) (Seconds) Oil-soluble part of shark's liver 500mg 13,800 21 Water-soluble part of shark's liver 50mg 9,500 Control 13,000 22 TABLE II Aqueous Extract of Bioassay(A) Bioassay(B) Shark's Gallbladder, Dosage (Units) (Seconds) Purified by: Charcoal adsorption 5mg XAD-2 adsorption Img 16 Anion-exchange resin adsorption 0.5mg 8,200 14 Purified active principle 0.15mg 9,600 Control 14,000 22 Standard bioassays referred to in the above description were performed as follows: Bioassay (A) Biological test for protective activity against ;II q 1 1 1 1 1 1 i i.l; i a WO 88/01274 PCT/AU87/00281 carbon tetrachloride (CC1 4 )-induced liver lesions in mice.

Male Std:ddy mice (weight 30-35g) were used in groups of 5 animals. Samples of test materials were administered orally 7 days at a suitable daily dose and 0.lml of 5% CC1 4 in olive oil was orally administered at 24hrs after the last sample administration. Blood was obtained from the orbital sinus at 24hrs after the CC14 administration. Serum was obtained by centrifugation (3,000 rpm., 10min) and.

glutamic pyruvic transaminase (GPT) activity was measured by Reitman-Frankel-Momose method. AcLivity was expressed as a comparison of GPT values between the sample-administered groups and controls.

Bioassay (B) Effect on respiration in nicotine administration to mice.

Male Std:ddy mice (weight 20-22g) were used in groups of 5 animals. Nicotine tartrate (3mg) was injected subcutaneously. Samples of test materials were orally administered 3hrs before nicotine administration. The time taken for 30 respirations was counted 5 minutes after nicotine administration.

Activity was expressed as a comparison of the counted time between the sample-administered groups and controls; The present invention also provides a pharmaceutical composition comprising an active substance as described above, together with a pharmaceutically acceptable carrier or diluent therefor. By way of example, the active substance can be formulated as stable tablets after being mixed as a ir WO 88/01274 PCT/AU87/00281 11 powder with a known carrier or bulking agent.

Alternatively, the active substance can be incorporated into a lotion or cream base for topical application. In yet another alternative, the active substance can for example be filled in soft gelatin capsules, if desired after being admixed with shark's liver oil. Such pharmaceutical compositions may be used, for example, for the protection of the liver or activation of liver function in the treatment of diseases or conditions affecting the liver such as hepatitis, nephritis, diabetes, etc.. Such compositions may also be used for the activation of regeneration of skin tissue, for example, in the treatment of dermatitis, trauma or acne.

Clinical tests which have been performed using compositions containing the active substance have specifically demonstrated its activity in restoration of the liver function, and in the treatment of seborrhea.

In a further aspect of this invention, there is provided a dietary or health food composition which comprises the active principle described herein, together with one or more appropriate base or carrier materials. Such a composition may, for example, be useful in the treatment of a hangover.

in another aspect, the present invention provides a cosmetic composition comprising the active principle as described above, together with a cosmetic base material.

The compositions of the present invention may also incorporate Known pharmaceuticals or other active ingredients, for example, antibiotics or other antibacterial substances.

WO 88/01274 PCT/AU87/00281 12 Further details of this invention will be apparent from the following Examples which illustrate the invention without limiting it in any way.

EXAMPLE 1 Preparation of Crude Active Principle of a mixture of liver and gallbladder isolated from 4kg of shark was homogenised in 300ml of water, and the mixture was centrifuged at 12,000 rpm for 30 minutes to obta.in a clear aqueous layer. of ion exchange resin of basic anion exchange type was added to the aqueous layer and the mixture was left to stand overnight. The resin was removed by filtration and washed with water. The resin was then extracted with 200ml of 0.5% sodium chloride solution. 100g of XAD2 was added to the extracted solution. XAD2 was removed by filtration and washed with water. XAD2 was extracted with 200ml of ethanol. From the extract, ethanol was removed by distillation to obtain 45mg of crude active powder.

EXAMPLE 2 Silica gel column chromatography 100g of crude active comLound obtained by adsorption on a XAD-2 column was subjected to chromatography on a silica gel column, using MeOH- CHC1 3

-H

2 0(30:70:6) as solvent, to afford white powder EXAMPLE 3 Thin layer chromatography (TLC) Crude active compound was subjected to TLC on a precoated silica gel 60 thin layer plate (Merck), using the system (parts by volume): n-BuOH(85)-AcOH(10)-H 2 0(5) and MeOH(40)-CHC13(60)- H20(10). The active principle showed as a single spot *7 r77 7.r WO 3801274 PCT/AU87/00281 13 on TLC, and was visualized by spraying with vanillin sulfuric acid reagent.

EXAMPLE 4 High performance liquid chromatography

(HPLC)

Final purification of crude active powder was achieved by successive application of preparative HPLC with a reverse phase column. 31g of the active compound in th° form of white powder, mp.140 0 was obtained from 100g of XAD-2 purified sample. The approximate representative retention time of the active compound was 16 minute. The conditions for HPLC were as follows: column: YMC-Pack A-324(ODS); flow rate: 20ml/min.; mobile phase: CH3CN-0.02N phosphate ammonium buffer (pH detector: refractive index.

EXAMPLE 5 Column chromatography on Sephadex Crude active compound (100g) obtained by adsorption on a XAD-2 column was subjected to gel filtration on Sephadex LH-20 column, using MeOH-CHC13(1:1) and then MeOH as eluents, to afford white powder (45g) from the MeOH fraction.

Rechromatography on the same column afforded 30g of almost pure white powder.

EXAMPLE 6 Gall-bladders (65g), obtained from 5 sharks of the species Rhizoprionodon acutus (ca 8Kg weight), were homogenized and then freeze-dried. This material (10.25g) was used as a source of the active principle, sodium scymnol sulphate. After defatting the material with refluxing n-hexane (100ml x3), it was extracted with methanol (100ml x3) under reflux for lh. The ,*7 j WO 88/0 1274 P CT/A U87/0028 I concentrate (3.67g) was dissolved in if120 (80m1) and applied to an Amberlite XAD-2 column (3.0 x 16.0cm).

rhe column was eluted with H 2 0 (400m1) and then with ,--hanol (400m1). Then, the ethanol eluate (1.95g) was appli.ed to Sephadex L11-20 column (3.0 x 32.0cm), chloroform and methanol After elution with chloroform and methanol (200m1), the column was developed with methanol in batches of 50m1.

Concentration of the methanol eluate containing the sodium salt gave a white gum (1.06g) Purification of this material (120ma) by HPLC yielded 85.6mg of sodium scymnol sulphate as white powder. The conditions for HPLC were as follows: column, YIIC-Pack A-324(ODS) lOx300mm; flow rate, 2m1/min; mobile phase, CH 3 CN-0.lN Sodium Phosphate Buffer (pH 6.43); detector, Refractive Index, Sodium scymnol sulphate has the following physical data: White powder; =21.75(0.5c, in MeOH); Anal.: Calcd. for C 27

H

47 0 9 SNa C;56.82 H;8.30 S;5.62 Na;4.03. Found: C;56.99 H;8.79 S;5.62 Na;4.23. SIMS mass 654[C7H 4SO. HN(C 2HO6 2, 574[C 27H 4706'(2H60)' IRv Kmaxcm- 1 3420, 2950, 1470, 1380, 1230, 1070, 980, 910,e 810. H-NMR (in CD 3 OD) 6(ppm): 4.22(1H,dd,J=4.5, 10.0Hz), 4.00 broad), 3.80(111, 3.45-3.30(11, in), 2.35-2.

in), 1.02(3H, d, J=6.2Hz),

S.

13 C-NMR (in CD 3

OD);

71.4 69.1 66.7 47.5(s), 43.1(d), 43.0(d), 36.5 3b'.9 35 .8 29.5(t), 28.8(t), 27 .9 13.1 m) 3 .80-3 .62 (3H, m), 15(2H, in), 2.05-1.02(23H1, 0 .92 (3H1, 0 .72 (3H1, (ppm): 74.1(d), 72.9(d), 61. 2(t) 48 .3 47 .8 41.0(d), 40.3(t), 37.0(d), 33.3(t), 32.1(t), 31.2(t), 24.3(t), 23.2(q), 18.1(q), 1-11-1-- 1- WO 88/01274 PCT/AU87/00281 EXAMPLE 7 Trials have been conducted using the active principle of this invent:on in an antiseborrheous lotion applied topically oy 40 male and female patients affected by long established (72) years facial hyperseborrhea. The trials were conducted as double blind trials with 20 patients applying a placebo and 20 patients applying the lotion containng the active principle.

In these trials, the treatment was applied three times daily (morning, midday and evening) over a period of 20 days, and an evaluation of seborrhea (Seborrhea Index) made at days 0, '(prior to treatment), 10 and 21, (at end of treatment).

The results showed a significantly greater improvement in the seborrhea for patients using the lotion containing the active principle than for patients using the placebo. It was also observed that this improvement was shown in both male and female patients.

'I

17J WO 8801274PCT/A U87/0028 I WO 88/01274 EXAMPLE 8 1. Cold Compositions cream Spermacetti Beeswax Carbopol. 934 Sodium Carbonate Rose water Rose oil Expressed almond oil Acta~ve principle Distilled water 6.Og 6 .O0g 10 .0g 4 5 .Om1 0 .0 2m1 56 .09 0 05 g 2 0 0g Tonic Ethanol 30m1 Active principle Flavour q.s.

Distilled water sufficient quantity to make 100mi, 7

Claims (12)

1. A compound of the general formula I, in substantially pure form, 120S03 wherein A is a cation.

2. A compound according to claim 1, wherein the cation is a sodium, potassium, calcium or ammonium ion, or an organic amine.

3. A method for the preparation of a compound of the general formula I as defined in claim 1, in substantially pure form, which comprises the steps of preparing an aqueous extract of the liver and/or gall-bladder of a shark, and isolating the said compound from said aqueous extract.

4. A method according to claim 3, wherein said step of isolation from the aqueous extract comprises at least one step selected from solvent extraction, adsorption and chromatography.

I lir;: i, ;I -I -i: 18 A pharmaceutical composition, comprising a compound of the general formula I as defined in claim 1, together with a pharmaceutically acceptable carrier or diluent therefor.

6. A pharmaceutical composition according to claim 5, in the form of a tablet, capsule, lotion or cream.

7. Use of a compound of the general formula I as defined in claim 1, for the protection of the liver or activation of liver function in the treatment of diseases or condition's affecting the liver.

8. A composition for the treatment of the skin C comprising a compound of the general formula I as *0Cdefined in claim 1, together with a topically acceptable carrier or diluent therefor.

9. A composition according to claim 8, further comprising an antibiotic or other antibacterial substance.

10. A cosmetic composition comprising a compound of the general formula I as defined in claim 1, together with a cosmetic base material.

11.Use of a compound of the general formula I as defined in claim 1, for the -treatment of the skin.

12. A compound as defin'.d in claim 1, a pharmaceutical or cosmetic composition comprising a said compound, or a method for the preparation or use thereof, substantially as hereinbefore described with reference to the Examples. Dated this 2nd day of February, 1990. J.M.BROADBENT and Y. KOSUGE, by their Patent Attorneys, DAVIES COLLISON FF%2' INTERNATIONAL SEARCH REPORT 101f40 101110011 AtofjcatiaA No PCT/AU 87/00281 1. CLASSIFICATION OF SUGJKCT MdATtN I Vatt.'Cation OV-00 1 1001,. Ad.ih* 1.1 A~CCorE#A to fflAtnl at 4411 Ctasuaig~alon (tpc) f o seemn National Ctaej.(ication am* IPC Int. Ci. 4 C07J 31/00, A61K 31/575 It. ICLOS 111AC)440 l.4.nsMuen OaCumentatien let~ Classificution Sritsm Classifcation symoe IPC C07J 31/00 us Cl. 260/397.2 cocunientation searchied Other then Moiimum Oocuntentation to thes [ateAt that suich 04oCUMAen ts r Included in@ Fie Ilds Searchedl AU IPC as above Ill. OOCUMVINTS CONS11010111 TO 31 RILIVANT' Category CItation Of Qocumelnt. 11 ,In nimacation. wee aecroortata. of the rolovanlt oalaogb II lieldvont to Cta1.1i 4o. 11 A US,A, 4296109 (LAURENT et al) 20 October 1981 (1) 10. 81) A USA, 3994878 (PARTRIDGE, Jr. et al) 30 November (1) 1976 (30.11.76) Sopoell) csagorsel of citse documents: is tater document~ DuC1li11110 afterf thS 111intarnetieat Along oate -A'dogffi~lldefnin IA 9014fll11111141 f 1114 ar whch 0 041 or ormenty seets and not An conflict witn the adolicatioh out A doumet dfinng he eneal eeteOf he lt hic ionet c-lgd to unforsta"IE the Pincialo or theory undatylln the considere to go of Oaltiscular raileeflc in% Mntlon 1 earlie document but pubefolene of *faft the Iteranationalf jeiuffel 0 oarvscular rieleanfes: thte claimed invenionA11 filing 4ole cannot of csildered novel of can,, .t gs ons~idered to 1.document wRich Mey' throw doubls on orienit, claimis? of invOlve en InV41ntgVI 11t1111 which 04 cited to6 eat1e66In@ helicatien 4ate of another degueon of IOMIA111utg re4flevnce the C11aim19d invention ci4tat Of 6101W gctal reason tao Iseotifseet COAnOt go C110116eer1141 to involve en inventive 0te0 unen theo -odocumient refernng to an eel1 dieclOllioaf. uae. OSAhiM1,14l of document #6CIPceneie -11 41ith Of or440 mee th0 Smith ee mean, MOMtS. such tafemetien going oteme to a Deosn &failed -1110 documenot Subhaheod grow to the letienelmi fltiig date but i 11tO4 GA. tater then the onoenty date claimed 196 doclument member of ie same saterit family IV. Ote of the Actual Comoltton of the international Search)te wi1 Mof ollti ef thts tnternatearnel Searech Roeort 17 November 1987 (17.11.87) 3a f~Ve;'Y'e )9?7 International Searching Authority A41V 41 vilathized OMCO Australian Patent Office HANSON Foeim $11IISA12tO iiecoi~t $hoot) Wsiery~ 104111 -~i T AhEX TO THE INTERNATIONAL SEARCH REPORT ON ITERNATINAL APPLICATION NO. PCT/AU 87/00281 This Annex lists the known publication level patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent Document Cited in Search Patent Family Members Report US 4296109 AU 51423/79 CA 1127630 DE 2843690 DK 4048/79 EP 10056 ES 484706 GB 2034715 JP 55051100 SU 818489 DE 2932166 US 3994878 AT 7513/76 AT 5464/79 AT 5463/79 AT 5465/79 BE 847131 CH 628907 CH 634337 DE 2645527 FR 2351998 FR 2407941 GB 1564806 GB 1564807 GB 1564808 GB 1564809 GB 1564810 IT 1068692 JP 52046061 NL 7611155 CH 626096 CH 626097 US 4038272 END OF ANNEX 7 Jr7-7g7 77-